Project Summary/Abstract The overarching goal of this proposal is to determine the structural basis for allosteric mechanisms governing gating and modulation in serotonin receptors (5-HT3AR). 5-HT3ARs play a crucial role in gastrointestinal functions, pain transmission, and mood disorders. Inhibitors of 5-HT3AR are in the clinical use to alleviate nausea and vomiting caused by cancer therapies, manage post-infection diarrhea and irritable bowel syndrome, and treat neurological conditions such as bipolar disorder and depression. Rational drug design has been limited by poor understanding of the structure-function correlations in 5-HT3AR and the downstream signaling events. We recently solved the structure of the full-length 5-HT3AR in the resting conformation by single-particle cryo- electron microscopy (cryo-EM). By capitalizing on this technical advancement and further biochemical optimization, we aim to determine the conformational changes underlying 5-HT3AR gating and lipid modulation. We will achieve these goals by a combination of multidisciplinary approaches that include cryo-EM, pulsed- EPR, and electrophysiology. Specifically, we will determine high-resolution snapshots of 5-HT3AR in multiple functional states, in modulator-bound conformations, and in the presence of membrane lipid constituents. These structures will be complemented with protein dynamic studies in a membrane environment and extensive functional analysis. Our proposed work is expected to provide structural blueprints of the channel in physiologically relevant conformations and unravel the molecular mechanisms underlying channel function. These studies will be foundational in the targeted drug design for safer and effective therapeutics.